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Time-course experiment

ABSTRACT: Buffy coat blood samples from 4 healthy donors were obtained from the Australian Red Cross Blood Service. The peripheral blood mononuclear cells were then extracted using a Ficoll Paque gradient. CD14 positive monocytes were then extracted using MACS (Miltenyi Biotec) magnetically labeled anti-CD14 antibodies. CD14 positive monocytes were then plated into 10cm petri dishes at a concentration of 1.5x107 cells per plate and differentiated into macrophages by addition of recombinant human CSF-1. Cells were treated with lipopolysaccharide (final conc. 10ng/ml in RPMI 1640 medium) for 2 or 6 hours and an untreated control had RPMI 1640 added only.

RNA was extracted using QIAGEN RNeasy columns as per the manufacturerM-^Rs protocol and analyzed by Nandrop and Bioanalyzer. RNA was further quality controlled with quantitative real-time PCR for known genes (TLR2, IL6 and IFNb). Samples that had the same expression pattern were then pooled from 4 individuals into 2 pools of two individuals. RNA was sent on dry ice to the Ramaciotti Centre for Gene Function Analysis, Sydney, Australia for processing and hybridization to Affymetrix Human Exon Arrays 1.0 as per the manufactererM-^Rs instructions. A ribo-minus step was included in the protocol. Resulting .CEL files were labeled AB01, AB02, AB03, AB04, AB05 and AB06. AB01 and 04 are the pooled untreated (0hour) time-points. AB02 and 05 are the samples treated for 2 hours, AB03 and 06 are samples treated for 6 hours.

ORGANISM(S): Homo sapiens

SUBMITTER: Anthony Beckhouse

PROVIDER: E-MEXP-2577 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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